Project Summary The goal of the proposed research is to understand the immunologic mechanisms by which specific major histocompatibility complex (MHC) class II molecules provide resistance to develop autoimmune type 1 diabetes (T1D) by evaluating insulin specific T cell responses, particularly focused on insulin B chain amino acids 9-23 (B:9-23). For many autoimmune disorders, including T1D, specific human leukocyte antigen (HLA) alleles confer disease risk as HLA-DQ8 and/or DQ2 are present in approximately 90% of all T1D patients. Strikingly, the polymorphic HLA-DQ6 (DQB*06:02) allele provides dominant protection from T1D. The objective of the proposed studies is to bridge the gap of knowledge regarding MHC class II disease resistance and self- antigen T cell response in T1D. Our long-term goal is to understand how insulin specific T cell responses can be manipulated for preventative and therapeutic purposes, ideally using mutated insulin B:9-23 as antigen specific therapy. The central hypothesis is that the MHC class II genotype shapes autoimmunity to insulin B:9- 23, determining susceptibility or protection from T1D. We plan to test our hypothesis using mutated insulin B:9- 23 peptides to determine T cell phenotypes in the context of MHC class II genotypes in murine and human diabetes. In specific aim 1, we will utilize a spontaneous mouse model of autoimmune diabetes including a model with a single amino acid mutation in the MHC class II beta chain (?57Ser?Asp) implicated in diabetes resistance. Insulin B:9-23 specific T cells restricted to risk and protective MHC class II molecules will be tracked using fluorescent tetramers. Experiments will be done to test the hypothesis that a protective MHC class II molecule leads to the generation of B:9-23 regulatory T cells (Tregs) that suppress islet autoimmunity; either through converting a pathogenic T cell to become regulatory and/or by differentiating B:9-23 specific Tregs restricted to the protective MHC class II molecule. In the second aim, we will study insulin and insulin B:9-23 specific T cell responses from well-characterized human subjects at risk for developing T1D and subjects without islet autoantibodies in a prospective longitudinal study. We now have the ability to measure both inflammatory and regulatory T cell responses to insulin with mutated B:9-23 peptides from the peripheral blood, which will allow for an increased understanding of disease pathogenesis in preclinical T1D. Further, healthy controls having the protective DQB*06:02 allele will be studied to isolate, characterize and T cell receptor (TCR) sequence B:9-23-specific Tregs, thereby creating reagents to assess the structural interactions of a diabetes resistant trimolecular complex, TCR-B:9-23-DQ6. A B:9-23/DQ6 tetramer will be produced to stain and phenotype ex vivo T cells from DQ6 individuals. The findings from this research are directly applicable to the design of insulin antigen specific therapies, monitoring and timing in the preclinical T1D period to prevent disease onset.